Direct Processing and Storage of Cell-Free Plasma Using Dried Plasma Spot Cards

Plasma separation cards represent a viable approach for expanding testing capabilities away from clinical settings by generating cell-free plasma with minimal user intervention. These devices typically comprise a basic structure of the plasma separation membrane, unconstrained porous collection pad, and utilize either (i) lateral or (ii) vertical fluidic pathways for separating plasma. Unfortunately, these configurations are highly susceptible to (i) inconsistent sampling volume due to differences in the patient hematocrit or (ii) severe contamination due to leakage of red blood cells or release of hemoglobin (i.e., hemolysis). Herein, we combine the enhanced sampling of our previously reported patterned dried blood spot cards with an assembly of porous separation materials to produce a patterned dried plasma spot card for direct processing and storage of cell-free plasma. Linking both vertical separation and lateral distribution of plasma yields discrete plasma collection zones that are spatially protected from potential contamination due to hemolysis and an inlet zone enriched with blood cells for additional testing. We evaluate the versatility of this card by quantitation of three classes of analytes and techniques including (i) the soluble transferrin receptor by enzyme-linked immunosorbent assay, (ii) potassium by inductively coupled plasma atomic emission spectroscopy, and (iii) 18S rRNA by reverse transcriptase quantitative polymerase chain reaction. We achieve quantitative recovery of each class of analyte with no statistically significant difference between dried and liquid reference samples. We anticipate that this sampling approach can be applied broadly to improve access to critical blood testing in resource-limited settings or at the point-of-care.


Measurement and Adjustment of the Hematocrit
We measured the initial hematocrit of the whole blood sample upon arrival from the vendor. We added 3 µL of whole blood to a 40-mm microhematocrit capillary tube and sealed the tube at one end with Critoseal putty. We centrifuged the microhematocrit capillary tubes at 1,200 RPM for 3 minutes using a ZipCombo centrifuge from LW Scientific. We obtained images of the microhematocrit tubes using an 8-bit EPSON Perfection V600 PHOTO scanner with a resolution of 800 DPI. We calculated the hematocrit of the sample by measuring the ratio of the length that RBCs occupied in the tube to the total sample length with ImageJ software. 1 We followed the same procedure for each sample for the measurement of hematocrit (N=2) We created samples of whole blood at different hematocrit values (20-60%) by adjusting the volume of native plasma in the sample. We confirmed the hematocrit value by measuring the hematocrit value as described above (N=2).

Fabrication of pDPS Cards
We used Adobe Illustrator to design the features of the card and printed the hydrophobic wax barriers using a previously reported double-sided transfer method. 2 Using this double-sided transfer method allows for unique features to be printed on the top and bottom of a single sheet of paper. First, we printed the top and bottom designs onto laminate sheets using a Xerox ColorQube 8580 wax printer. Next, we aligned a sheet of chromatography paper with the top and bottom designs using a custom acrylic alignment jig. Finally, we used a Promo Heat CS-15 Tshirt press (45 seconds at 280 °F) to transfer the wax from the laminate sheets to the paper to form hydrophobic barriers through the full thickness of the paper. These barriers control sample flow and distribution in pDPS cards. We cut all adhesive and laminate layers using a Boss laser cutter. We manually cut the PSM and Leukosorb membranes cut using a hammer-driven circular metal punch (10-mm diameter).

Estimation of Output Plasma Volume by Quantitation of Hemoglobin
We estimated the volume of plasma contained in each 6-mm diameter paper punch generated with pDPS cards by quantitation of hemoglobin using Drabkin's reagent and a standard protocol. 3 We constructed individual calibration curves using lyophilized hemoglobin standards rehydrated in ACS reagent grade (ASTM Type 1) water over a range of 3-18 g/dL. We varied the input sample volume of hemoglobin standard used for each calibration curve (3- Blank samples comprised deionized water. For elution studies, we followed the above protocol with the following change: we varied the concentration of surfactant (final concentration 0.05% v/v) in phosphate buffered saline instead of using the supplied diluent. We investigated four surfactants (Tween 20, Brij 35, CHEMAL LA-9, and surfactant 10G) and EDTA for achieving optimal elution of transferrin receptor protein.

Preparation of pDPS Samples for the Quantitation of Potassium by ICP-AES
We applied 75 µL of venous whole blood to pDPS cards and allowed them to dry overnight.
We pooled two punches (6-mm diameter) from each pDPS card and prepared liquid plasma reference samples via centrifugation. We added both samples (pDPS punches and liquid reference) to individual 5 mL round bottom flasks and digested the samples in 3 mL nitric acid (70% v/v) at 100 °C for 90 minutes. Following digestion, we evaporated the remaining nitric acid from the round bottom flasks. Once cooled, we reconstituted the samples in 3 mL nitric acid (10% v/v) and transferred the contents to 5 mL Eppendorf tubes. We prepared blank samples with two punches (6-mm diameter) of unpatterned TFN and followed the digestion procedure above. We also eluted pooled paper punch samples from pDPS cards using 3 mL ACS reagent grade (ASTM S-6 Type 1) water in 5 mL Eppendorf tubes at 4 °C overnight (ca. 16 hours). We calibrated the ICP-AES with liquid calibrants (0.3-10 ppm potassium) before each experimental run. We analyzed all digested and eluted samples at a wavelength of 766.5 nm using a single phase, high dispersion Prodigy Spec ICP-AES by Leeman Labs Inc. (Hudson, NH).

Preparation of samples for DNA extraction
We applied 75 µL of venous whole blood to 6 pDPS cards and allowed them to dry overnight. After drying, we peeled off the adhesive layers that kept Leukosorb and PSM membranes adhered to TFN, and we punched them out of the adhesive layers using a hammerdriven circular metal punch (11-mm diameter). We transferred the membranes (Leukosorb and PSM) to a 2-mL centrifuge tube, making sure the membranes touched the bottom of the tubes.
We performed the extraction accordingly to the manufacturer's instructions, with no deviations from the protocol. 5 For whole blood samples (liquid reference), we added 75 µL of venous whole blood to 6 2-mL centrifuge tubes and performed the extraction following the manufacturer's instructions (QIAamp® DNA Mini and Blood Mini Handbook), with no deviations from the protocol.
We added 125 µL of PBS 1X buffer to bring the sample up to the required 200 µL.

qPCR amplification of DNA extracts and gel electrophoresis
We used an Applied Biosystems QuantStudio 3 Real-Time PCR System to perform quantitative PCR (qPCR). We set reaction volumes as 50 µL/well, using the SuperScript™ III Platinum SYBR Green One-Step RT-qPCR Kit. Each reaction contained 16 µL of Nuclease-Free (NF) water, 25 µL of 2x SYBR green reaction mix, 1 µL of Superscribe III Taq Mix, 2 µL of Reverse and Forward primer mixture (5 µM in each primer,

S-7
We set the amplification profile as a single cycle of enzyme activation at 95 °C for 20 s, followed by 40 cycles of denaturation at 95 °C for 1 s and annealing at 60 °C for 20 s, with single fluorescence acquisition. We set the melt curve profile as a single cycle at 95 °C for 1 s, followed by an annealing step at 60 °C for 20 s and a dissociation step at 95 °C for 1 s, with single fluorescence acquisition.
After amplification, we analyzed PCR products using a Lonza FlashGel System. The analysis was performed as per manufacturer's instructions. Briefly, we hydrated the wells using 500 µL of NF water, adding the water in the first well and tilting the plate, so excess water would flow from one well to the next. Next, we added 4 µL of 5X loading dye to 10 µL of sample and mixed it thoroughly by pipetting. We carefully added 4 µL of the stained sample to each well of a Lonza 1.2% Agarose gel, and 4 µL of DNA ladder markers to both outer wells. We ran the separation at 250 V for 6 min, using a BioRad PowerPac HC power supply, using a Lonza camera and dock system. We recorded the gel image after 6 min.
S-8 Liquid plasma was produced as a reference sample via centrifugation.